Limits...
Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum.Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Show MeSH
Delivery of TALE transcriptional activators into cultured human cells. (a) Design of an 18.5-repeat TALE activator fused C-terminally to a VP64 activation domain and N-terminally to (−30)GFP and an NLS. The overall net theoretical charge of the fusion is −43. (b) Activation of NTF3 transcription by traditional transfection of plasmids encoding TALE-VP64 activators that target sites in the NTF3 gene, or by RNAiMAX cationic lipid-mediated delivery of the corresponding NTF3-targeting (−30)GFP-TALE-VP64 proteins. For protein delivery experiments, 25 nM VEGF TALE, 25 nM NTF3 TALE 1, or 25 nM NTF3 TALEs 1–5 (5 nM each) were delivered with 1.5 µL RNAiMAX in 275 µL DMEM-FBS without antibiotics for 4 hours before being harvested. For plasmid transfections, a total of 300 ng of one or all five NTF3 TALE expression plasmids (60 ng each) were transfected with 0.8 µL Lipofectamine 2000 in 275 µL DMEM-FBS without antibiotics and harvested 48 hours later. Gene expression levels of harvested cells were measured by qRT-PCR and are normalized to GAPDH expression levels. Incubation times for TALE activators by plasmid transfection and protein delivery were those found to give maximal increases in NTF3 mRNA levels. (c) Time course of TALE activation for protein delivery and plasmid transfection by measuring NTF3 mRNA levels and then normalizing each method to the highest activation value achieved over any time point for that method. Optimal protein (25–50 nM) and lipid dosage (1.5 µL RNAiMAX) was used for each delivery technique. Error bars reflect s.d. from three biological replicates performed on different days.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4289409&req=5

Figure 3: Delivery of TALE transcriptional activators into cultured human cells. (a) Design of an 18.5-repeat TALE activator fused C-terminally to a VP64 activation domain and N-terminally to (−30)GFP and an NLS. The overall net theoretical charge of the fusion is −43. (b) Activation of NTF3 transcription by traditional transfection of plasmids encoding TALE-VP64 activators that target sites in the NTF3 gene, or by RNAiMAX cationic lipid-mediated delivery of the corresponding NTF3-targeting (−30)GFP-TALE-VP64 proteins. For protein delivery experiments, 25 nM VEGF TALE, 25 nM NTF3 TALE 1, or 25 nM NTF3 TALEs 1–5 (5 nM each) were delivered with 1.5 µL RNAiMAX in 275 µL DMEM-FBS without antibiotics for 4 hours before being harvested. For plasmid transfections, a total of 300 ng of one or all five NTF3 TALE expression plasmids (60 ng each) were transfected with 0.8 µL Lipofectamine 2000 in 275 µL DMEM-FBS without antibiotics and harvested 48 hours later. Gene expression levels of harvested cells were measured by qRT-PCR and are normalized to GAPDH expression levels. Incubation times for TALE activators by plasmid transfection and protein delivery were those found to give maximal increases in NTF3 mRNA levels. (c) Time course of TALE activation for protein delivery and plasmid transfection by measuring NTF3 mRNA levels and then normalizing each method to the highest activation value achieved over any time point for that method. Optimal protein (25–50 nM) and lipid dosage (1.5 µL RNAiMAX) was used for each delivery technique. Error bars reflect s.d. from three biological replicates performed on different days.

Mentions: Next we tested lipid-mediated delivery of TALE-VP64 transcriptional activators into HEK293T cells. While modestly effective cleavage of endogenous genes by delivered TALEN proteins has been demonstrated in mammalian cells in the absence of serum using cationic peptides,29 the delivery of TALE-based transcription factor proteins has not yet been reported, and no effective delivery of TALE proteins in serum-containing media has been previously described to our knowledge. We targeted the gene for neurotrophin-3 (NTF3), a neural growth factor that when mutated has been associated with neurodegenerative diseases and hearing loss.30 We fused a previously described NTF3-targetting TALE-VP6431 to (−30)GFP (Fig. 3a) and treated HEK293T cells with 25 nM (−30)GFP-NTF3 TALE1-VP64 and 1.5 µL RNAiMAX under the conditions optimized for Cre delivery. Gene expression levels of NTF3 4 hours after treatment were 3.5-fold higher in cells treated with 25 nM (−30)GFP-NTF3 TALE1-VP64 and RNAiMAX than in untreated cells, cells treated with RNAiMAX only, or cells treated with a VEGF-targeting TALE transcriptional activator (Fig. 3b). Similar levels of NTF3 expression were observed 48 hours after transfection of plasmids encoding the same NTF3-targeting TALE-VP64 (Fig. 3b).


Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

Delivery of TALE transcriptional activators into cultured human cells. (a) Design of an 18.5-repeat TALE activator fused C-terminally to a VP64 activation domain and N-terminally to (−30)GFP and an NLS. The overall net theoretical charge of the fusion is −43. (b) Activation of NTF3 transcription by traditional transfection of plasmids encoding TALE-VP64 activators that target sites in the NTF3 gene, or by RNAiMAX cationic lipid-mediated delivery of the corresponding NTF3-targeting (−30)GFP-TALE-VP64 proteins. For protein delivery experiments, 25 nM VEGF TALE, 25 nM NTF3 TALE 1, or 25 nM NTF3 TALEs 1–5 (5 nM each) were delivered with 1.5 µL RNAiMAX in 275 µL DMEM-FBS without antibiotics for 4 hours before being harvested. For plasmid transfections, a total of 300 ng of one or all five NTF3 TALE expression plasmids (60 ng each) were transfected with 0.8 µL Lipofectamine 2000 in 275 µL DMEM-FBS without antibiotics and harvested 48 hours later. Gene expression levels of harvested cells were measured by qRT-PCR and are normalized to GAPDH expression levels. Incubation times for TALE activators by plasmid transfection and protein delivery were those found to give maximal increases in NTF3 mRNA levels. (c) Time course of TALE activation for protein delivery and plasmid transfection by measuring NTF3 mRNA levels and then normalizing each method to the highest activation value achieved over any time point for that method. Optimal protein (25–50 nM) and lipid dosage (1.5 µL RNAiMAX) was used for each delivery technique. Error bars reflect s.d. from three biological replicates performed on different days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4289409&req=5

Figure 3: Delivery of TALE transcriptional activators into cultured human cells. (a) Design of an 18.5-repeat TALE activator fused C-terminally to a VP64 activation domain and N-terminally to (−30)GFP and an NLS. The overall net theoretical charge of the fusion is −43. (b) Activation of NTF3 transcription by traditional transfection of plasmids encoding TALE-VP64 activators that target sites in the NTF3 gene, or by RNAiMAX cationic lipid-mediated delivery of the corresponding NTF3-targeting (−30)GFP-TALE-VP64 proteins. For protein delivery experiments, 25 nM VEGF TALE, 25 nM NTF3 TALE 1, or 25 nM NTF3 TALEs 1–5 (5 nM each) were delivered with 1.5 µL RNAiMAX in 275 µL DMEM-FBS without antibiotics for 4 hours before being harvested. For plasmid transfections, a total of 300 ng of one or all five NTF3 TALE expression plasmids (60 ng each) were transfected with 0.8 µL Lipofectamine 2000 in 275 µL DMEM-FBS without antibiotics and harvested 48 hours later. Gene expression levels of harvested cells were measured by qRT-PCR and are normalized to GAPDH expression levels. Incubation times for TALE activators by plasmid transfection and protein delivery were those found to give maximal increases in NTF3 mRNA levels. (c) Time course of TALE activation for protein delivery and plasmid transfection by measuring NTF3 mRNA levels and then normalizing each method to the highest activation value achieved over any time point for that method. Optimal protein (25–50 nM) and lipid dosage (1.5 µL RNAiMAX) was used for each delivery technique. Error bars reflect s.d. from three biological replicates performed on different days.
Mentions: Next we tested lipid-mediated delivery of TALE-VP64 transcriptional activators into HEK293T cells. While modestly effective cleavage of endogenous genes by delivered TALEN proteins has been demonstrated in mammalian cells in the absence of serum using cationic peptides,29 the delivery of TALE-based transcription factor proteins has not yet been reported, and no effective delivery of TALE proteins in serum-containing media has been previously described to our knowledge. We targeted the gene for neurotrophin-3 (NTF3), a neural growth factor that when mutated has been associated with neurodegenerative diseases and hearing loss.30 We fused a previously described NTF3-targetting TALE-VP6431 to (−30)GFP (Fig. 3a) and treated HEK293T cells with 25 nM (−30)GFP-NTF3 TALE1-VP64 and 1.5 µL RNAiMAX under the conditions optimized for Cre delivery. Gene expression levels of NTF3 4 hours after treatment were 3.5-fold higher in cells treated with 25 nM (−30)GFP-NTF3 TALE1-VP64 and RNAiMAX than in untreated cells, cells treated with RNAiMAX only, or cells treated with a VEGF-targeting TALE transcriptional activator (Fig. 3b). Similar levels of NTF3 expression were observed 48 hours after transfection of plasmids encoding the same NTF3-targeting TALE-VP64 (Fig. 3b).

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum.Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Show MeSH